The innate immune system recognizes microbial pathogens using germ line encoded receptors which recognize conserved structures made by microbes. For example, the pattern recognition receptor TLR4 recognizes lipopolysaccharides produced as part of the cell walls of Gram-negative bacteria. When professional phagocytic cells such as macrophages and dendritic cells internalize and degrade microbes, structures may be released. For example, bacterial cell wall peptidoglycan may be cleaved to release muramyl dipeptide, a ligand for the cytosolic pattern recognition receptor Nod2. In this project, we will stretch the definition of a pattern recognition receptor to include the host enzyme hexokinase, a central enzyme in cellular glucose metabolism. We have shown that upon degradation of peptidoglycan in phagosomes a component sugar, N-acetylglucosamine, is freed up and transported into the cytosol where it interacts with hexokinase causing release of the enzyme from the mitochondrial surface. Hexokinase release from mitochondria initiates a signaling process culminating in activation of caspase-1 in an inflammasome complex which is responsible for processing and release of the key pro-inflammatory cytokines IL-1 and IL-18 (among other effects). Thus hexokinase is the receptor that detects the presence of this microbe-derived sugar. Endogenously produced N-acetylglucosamine is not normally found free in the cytosol and thus does not engage hexokinase. In this project we will characterize this innate immune mechanism further in three aims. In aim one we will characterize the cell biology of hexokinase signaling in macrophages and dendritic cells and determine how the process is regulated so as not to be toxic to the cells. In aim two we will define the role of the pathway in host defense through characterizing the function of the host enzyme N-acetylglucosamine kinase as a negative regulator in vitro and in vivo in knockout mice. In aim three well will explor how the Gram-positive peptidoglycan recycling system can be manipulated to influence signaling by this pathway in immune cells and alter microbial pathogenesis. PUBLIC HEALTH RELEVANCE: Growing evidence suggests a strong relationship between inflammation and metabolic diseases such as obesity, type 2 diabetes, metabolic syndrome, and heart disease, although the ways in which inflammatory signaling and metabolic processes might be related are unclear. In this project, we are characterizing a new inflammatory signaling pathway that makes use of immune cells' basic metabolic enzymes to detect bacteria and trigger release of inflammatory mediators.